Telomere integrity is essential for cell survival and proliferation. Accordingly, dysfunctional telomeres can initiate genomic instability, cellular senescence, and organismal aging. Telomere structure is provided by binding of shelterin to telomeric DNA repeats forming a nucleoprotein complex. Similar to the human shelterin, in fission yeast, Schizosaccharomyces pombe, shelterin is composed of telomeric sequence-specific double-stranded and single-stranded DNA binding proteins, Taz1 and Pot1, respectively, accompanied by their protein interaction partners, Rap1, Poz1, and Tpz1, which form a protein bridge connecting Taz1 and Pot1. Shelterin protects telomere integrity as well as regulates telomerase. With the parent grant support, we biochemically identified Tpz1 mutants that can individually but specifically disrupt its interactions with Poz1, Ccq1, or Pot1. Using these separation-of-function mutants of Tpz1, we found that the complete linkage between telomere dsDNA and ssDNA binding proteins within the shelterin complex is required for defining the telomerase-nonextendible state of telomeres. Moreover, epistasis analyses revealed that Tpz1 also participates in the activation of telomeres to the extendible state via its interaction with Ccq1. Our results suggest critical regulatory roles of Tpz1 in the telomere ON/OFF binary switch. Here, we request supplemental funds to use emerging technologies and interdisciplinary teams to further advance our mechanistic understanding of shelterin to a new level. The two aims are: (1) Identify shelterin interactomes and post-translational modifications on shelterin components using chemical cross-linking coupled with mass spectrometry analysis (CXMS); (2) Reveal the functional roles of Tpz1 in coordinating shelterin components for telomerase-mediated and recombination-based telomere elongation via analyzing telomere sequences using single molecule, real-time (SMRT) next- generation sequencing technology.